Process for preparing protease from endothia parasitica using glucanases to reduce viscosity

ABSTRACT

The present invention relates to a process for preparing protease from Endothia parasitica, wherein a glucanase or a mixture of glucanases acting on the viscosity increasing polysaccharides co-produced by the fungus, is introduced in the medium during or after fermentation.

The present invention relates to a process for preparing the protease which is secreted by Endothia parasitica and which coagulates the milk.

The preparation of this protease is already described in French Patent No. 1,401,474.

It has now been found that the viscosity of the fermentation medium increases considerably during the protease production stage, which complicates the operations of purification and isolation and more particularly ultrafiltration, and that the viscosity was particularly due to the presence of a polysaccharide co-produced by the microorganism together with protease.

The process according to the invention consists in introducing in the fermentation medium an enzyme which acts on the polysaccharide co-produced by the microorganism.

The enzyme may be introduced either at the beginning of the fermentation, or during the fermentation, or only during the final treatment of the fermentation medium, before filtration of the biomass or during concentration of the filtrate by ultrafiltration or microfiltration.

It is also possible to introduce different enzymes in the medium, either simultaneously or separately at different stages in the process.

Despite the presence of protease in the medium, the enzyme acts on the polysaccharide and reduces the viscosity of said medium, whether during the fermentation or after said fermentation was stopped.

In the process for preparing a coagulating protease by fermentation of Endothia parasitica, it is possible to use glucanases such as the beta-1,3 glucanase of Basidiomycetes described in U.S. Pat. No. 3,423,288, the beta-1,3 beta-1,6 glucanases, such as that marketed by NOVO under the trademark Glucanex or that marketed by Gist Brocades under the trademark Rapidase GL 150, or also fungal alpha-amylases, such as Fungamyl marketed by NOVO, or Amylase P200 marketed by Gist Brocades.

Generally, the enzyme will be introduced in the fermentation medium only after a culture period higher than 50% of the total duration estimated for a fermentation without enzyme, and preferably higher than 75%; under these conditions, the viscosity of the medium remains limited and the production of protease is not reduced and can even be improved; it is indeed found that the viscosity of the broth at the end of the operation is clearly less than that of a fermentation without enzyme, about 2 to 4 times less, without any reduction of the biomass.

The filtrate, which in this case is obtained after separation of the cells, is less viscous and can be concentrated by ultrafiltration in a conventional device up to a concentration higher than 30 g/l of protease and even up to 40 g/l, whereas after a conventional fermentation, the maximum concentration is only about 10 g/l; in the end, the final filtration on a low porosity membrane becomes easier.

According to another aspect, the process of the invention consists in treating the fermentation broths before filtration, for 1 to 5 hours, with 50 mg/l to 1 g/l of an enzyme composition active on the polysaccharide; the viscosity of the broth is reduced by 2 to 5 times as well as the filtering time required for separating the biomass from the medium containing the desired product.

According to a last aspect, the process of the invention consists in treating the filtrate obtained after separation of the cells, with an enzyme which hydrolyzes the polysaccharide co-produced during the concentration of this filtrate by ultrafiltration, in order to obtain a solution with a high concentration of the desired product when said product cannot be isolated directly by precipitation or evaporation of the solvent without being denaturated; in such a case, an alpha-amylase is preferably used.

The following is a description of examples of embodiment of the invention, applied to the fermentation of Endothia parasitica.

The inoculums are prepared from frozen spores of Endothia parasitica, strain ATCC 14729, by culture at 28° C. in a sterilized aqueous medium containing glucose, soya flour, mineral salts, and autolytic yeast extracts.

Production is carried out at 28° C. in a sterile aqueous medium of the same type as the previous one.

Viscosities are measured with a Brookfield viscometer; the coagulating activities were determined by the method recommended by the Federation Internationale de Laiterie (International Milk Federation) and published in the Official Journal of the French Republic of Mar. 20, 1981.

EXAMPLE 1 Enzymatic Treatments During Fermentation

The tests were conducted with Glucanex or Amylase P 200 introduced at different moments of the fermentation, total duration of said fermentation being about 90 hours. Amylase P 200 contains a fungal alpha-amylase of Aspergillus oryzae and is in powder form, of FAU titer : 4540/g, one FAU corresponding to the quantity of enzyme hydrolyzing 5.26 g of soluble starch in one hour, at 37° C. and pH 4.7.

Glucanex is a powder containing an enzyme secreted by a Trichoderma strain; it contains 300 units of beta-glucanase per gramme.

The added quantities of enzyme, the addition times as well as the viscosity of the fermentation medium and the coagulating activity of the concentrate are indicated in Table I hereafter.

                                      TABLE I                                      __________________________________________________________________________                                                   CONCENTRATE                                  ENZYME            VISCOSITIES OF MEDIUM                                                                          COAGULATING                                                                              CENTRATE               TEST        CONCENTRATION                                                                             ADDITION                                                                              (mPa.s)         ACTIVITY  VISCOSITY              No  ENZYME  (mg/l)     TIME   40 hr                                                                             47 hr                                                                             64 hr                                                                             72 hr                                                                             88 hr                                                                              g/l       (mPa.s)                __________________________________________________________________________     1   Glucanex                                                                               20          0              1000                                                                              1140                                                                               15                               2   "       20         42               950                                                                              680 28                               3   Control  0                1900                                                                              2600                                                                              3750                                                                              3700                                                                              2200                                                                               8.2       93                     4   Glucanex                                                                               20         65     1900                                                                              2500                                                                              3050                                                                              1600                                                                              890 30        8                      5   "       50         65        2250                                                                              2700                                                                               750                                                                              420 41        9                      6   "       100        65        2250                                                                              2700                                                                               400                                                                              380 43        9                      7   Control  0                1100                                                                              1700                                                                              -- 1900                                                                              1000                                 8   Glucanex                                                                               10         45 and 68                                                                              950                                                                              1300                                                                              -- 1100                                                                              650                                  9   Control  0                 720                                                                               920                                                                               640                                                                              -- 800                                  10  Glucanex                                                                               200        23      240                                                                               240                                                                                106                                                                             -- 400                                  11  Amylase P200                                                                           20         45 and 65                                                                             1300                                                                               800                                                                               820                                                                              1000                                                                              850                                  12  "       40         65     1300                                                                              1250                                                                              1200                                                                               900                                    13  Control  0          0     1500                                                                              1900                                                                              2200                                                                              2800                                                                              2100                                 14  Glucanex                                                                               42         72     1900                                                                              1800                                                                              1600                                                                              2700                                                                              900                                  __________________________________________________________________________

The broths in tests 13 and 14 were treated after 90 hours of fermentation: filtration through canvas to separate mycelium, followed by concentration of the filtrate by ultrafiltration with a membrane permeable to compounds of molecular weight less than about 10000.

The results obtained are shown in Table II below.

Test 13 was conducted in the conventional way without adding any enzyme; in test 14, 40 mg of Glucanex were added per liter of broth after 72 hours of fermentation.

                                      TABLE II                                     __________________________________________________________________________                                                  CONCENTRATE                                                                              CON-                    TEST                                                                               BROTH VISCOS-                                                                            WEIGHT OF                                                                              FILTRATION                                                                             WEIGHT OF                                                                              FILTRATE                                                                              COAGULATING                                                                              CENTRATE                N°                                                                          ITY (mPa.s)                                                                              BROTH   TIME    MYCELIUM                                                                               VISCOSITY                                                                             ACTIVITY  VISCOSITY               __________________________________________________________________________     13  1600      158 kg  24 min  31 kg   5      10 g/l    120 mPa.s               14   900      142 kg  19 min  30.5 kg 4      38 g/l    120                     __________________________________________________________________________                                                            mPa.s              

EXAMPLE 2 Enzymatic Treatments of the Broth at End of Fermentation

The broths were treated after 88 hours of culture; they contained about 7.5 g/kg of polysaccharide and had a viscosity of 1000 mPa.s. 200 mg/l of various enzymatic compositions in powder form or 10 ml/l of liquid enzymatic compositions were added at this stage.

The results obtained for various contact times at room temperature are shown in Table III hereunder.

                                      TABLE III                                    __________________________________________________________________________                                          BROTH VISCOSITY (mPa.s) after             ENZYMATIC COMPOSITION                                                                          SUPPLIER                                                                              ENZYMATIC ACTIVITY                                                                           0.5 hr                                                                              1 hr                                                                               2 hr                                                                               5 hr                         __________________________________________________________________________     Hemicellulase Reg 2                                                                            Gist-Brocades                                                                         Pectinase + galacta-                                                                         920  770 520 550                                                 nase + galactomannase                                   Fungamyl        Novo   Alpha-amylase 690  550 550 450                          Amylase P 200   Gist-Brocades                                                                         Alpha-amylase 850  600 680 550                          Glucanex        Novo   Beta-1,3 beta-1,6                                                                            650  420 280 150                                                 glucanase                                               Rapidase GL 150 Gist-Brocades                                                                         Beta-1,3 beta-1,6                                                                            850  600 520 480                                                 glucanase                                               finizym         Novo   Beta-1,3 beta-1,4                                                                            920  900 880 850                                                 glucanase                                               __________________________________________________________________________

EXAMPLE 3 Enzymatic Treatments of the Fermentation Filtrate after Separation of the Mycelium

The enzyme was added before the beginning of the ultrafiltration. Fungamyl is a liquid composition of fungal alpha-amylase of titer 800 FAU/g; Maxamyl and BAIF, marketed by Gist-Brocades, are bacterial amylases.

The Table IV shows the drop of viscosity with time of a pre-concentrated filtrate in which various enzymes have been introduced in variable quantities.

The filtrate, preconcentrated by ultrafiltration, and having a pH of 4.3, had a viscosity of 70 mPa.s; the quantities of enzymatic compositions are given in g for 100 g of protease in the preconcentrated filtrate.

                                      TABLE IV                                     __________________________________________________________________________                     QUANTITY                                                                              VISCOSITY (mPa.s) after                                 ENZYMATIC COMPOSITION                                                                          g/100 g                                                                               25 mins.                                                                            50 mins.                                                                            100 mins.                                                                           150 mins.                                                                           175 mins.                           __________________________________________________________________________     Maxamyl         4.76   58   46   38   32                                       BAIF            3.8    46   32   26   24                                       Amylase P200    4.76   5     5    5    5   5                                                   0.95   7.5   5    5    5   5                                                   0.095  22   18   16   15   14                                  Fungamyl        4.76   10   10   10                                                            0.095  28   20   16                                            Glucanex        0.95   63   62   63   62                                       Dextranase Novo 4.76   70   68        65                                       Pectinase SP 249 Novo                                                                          4.76   70   68        65                                       __________________________________________________________________________

EXAMPLE 4 Enzymatic treatments during concentration of the filtrate

The whole process was carried out in the conventional way: fermentation, separation of the biomass by filtration and concentration of the filtrate by ultrafiltration.

This last operation was discontinued when the flow had become virtually nil. The concentrate was then found to be of pH 3.9 and to contain 9 g/l of protease.

40 mg of amylase P 200 or 40 mg of Fungamyl were introduced for 10 kg of concentrate and ultrafiltration was resumed after leaving the mixtures to stand for 2 hours and 15 minutes at room temperature.

After 45 minutes of concentration, the concentrate obtained had a protease concentration of 45 g/l in the case of the treatment with amylase P 200, and 42 g/l in the case of the treatment with Fungamyl. 

What is claimed is:
 1. In a process for preparing a protease, comprising the steps of culturing the fungus Endothia parasitica in a suitable fermentation medium, separating the cells from the medium, concentrating the resultant medium, and isolating from the concentrated medium a protease produced by the fungus,the improvement comprising reducing increased viscosity caused by a polysaccharide or polysaccharides produced by Endothia parasitica during the fermentation process by adding to said medium an enzyme selected from the group consisting of a beta-1,3 glucanase, a beta-1,3-beta-1,6 glucanase or an alpha-amylase, or a mixture thereof wherein said enzyme or mixture of enzymes is introduced during or after fermentation, but prior to isolation of said protease.
 2. A process as claimed in claim 1, wherein said enzyme or mixture of enzymes is introduced into said medium during fermentation.
 3. A process as claimed in claim 1, wherein said enzyme or mixture of enzymes is introduced into said medium at the end of fermentation but before the separation of the cells.
 4. A process as claimed in claim 1, wherein said enzyme or mixture of enzymes is introduced into said medium after the separation of the cells but before concentration of the medium by ultrafiltration or microfiltration.
 5. A process as claimed in claim 1 wherein said enzyme or mixture of enzymes is introduced into said medium during concentration by ultra-filtration.
 6. A process as claimed in claim 1, wherein said enzyme is a beta-1,3 beta-1,6 glucanase, which is introduced into said medium during or at the end of fermentation.
 7. A process as claimed in claim 1 wherein said enzyme is an alpha-amylase and said alpha-amylase is introduced into said medium during concentration. 